B cell regulation of lung neutrophils during fungal pneumonia. (A–C) Lung intravital microscopy was performed in mice infected with A. fumigatus (24 h), and neutrophils were imaged and quantified. Scale bars, 100 µm. (B) C57BL/6 mice were compared with Ighm^−/− mice with and without LXA₄ administration (5 µg i.v. at the time of infection). (C) C57BL/6 mice were compared with Alox15^−/− mice. A–C represent 3–4 independent experiments using 14 total mice. (D) Neutrophil (i.v. fluorescently conjugated anti-Ly6G mAb, red) and B cell (Cd19^Zsgreen1, blue) physical interactions were observed using lung intravital microscopy and highlighted as colocalization (white in the right panel). Scale bars, 70 µm. (E) Untreated Cd19^Zsgreen1 mice were compared with CXCL13 i.t. pretreated mice. (F and G) B cell neutrophil interactions were quantified and compared between Cd19^Zsgreen1 mice treated with i.v. isotype control antibody versus a neutralizing anti-CXCL13 mAb (F) or between C57BL/6 versus Cxcr5^−/− mice (G). (H) The acquisition of MHCII from B cells to neutrophils and the initiation of neutrophil apoptosis using annexin V staining was evaluated using flow cytometry. (I) MHCII⁺ annexin V⁺ lung neutrophils were quantified by flow cytometry and compared between control and exogenous CXCL13 i.t. treated mice. For imaging experiments, D–G pooled FOV replicates are shown, and n = 3 independent experiments were performed per condition using a total of 18 mice. In H and I, n = 5 independent experiments using 10 mice in total. Exact P values were determined using Student’s t test to compare two groups or ANOVA to compare three groups. Pooled data are presented as mean ± SD. *, P < 0.05; **, P < 0.01.